In this study we investigated the expression patterns of Hsp90α and Hsp90β of Paralichthys olivaceus after the temperature treatment and Vibrio anguillarum infection. qRT-PCR was used to detect the expression of Hsp90 in different tissues of P. olivaceus during a series of growth stages (1-, 8-, 12-, and 24-month old). The results showed that Hsp90α and Hsp90β were expressed in 13 tissues including liver, spleen, head kidney, kidney, heart, muscle, dorsal skin, stomach, intestine, gill, pelvic fin, brain, and blood at all growth stages. The expression level of Hsp90β was overall higher than that of Hsp90α. Expression of Hsp90α was significantly higher at 8-month than at other growth stages in the liver, gills, dorsal skin, dorsal muscle, and pelvic. As the level of Hsp90β varied at different growth stages, it was observed to be higher in the liver and intestine. After a treatment at 5–32℃ for 1 h, the expression of Hsp90α in the liver and spleen was rapidly increased, whereas the expression of Hsp90β did not changed evidently. After a continuous treatment at 10℃ and 28℃ for 8 h, the expression of both Hsp90α and Hsp90β in the liver and spleen declined after an initial increase. Expression of Hsp90α varied in a wider range than Hsp90β. Within 72 h after the infection with V. anguillarum, expression of Hsp90α and Hsp90β in the spleen first rose and then decreased, and expression of Hsp90β had a larger variation range. These results indicated that Hsp90α and Hsp90β might have distinct expression patterns in different tissues at various growth stages. Hsp90α may be apt to respond to high temperature, while Hsp90β could be more sensitive to pathogen infection. These data will provide the basic data for better understanding of the functions of Hsp90 in P. olivaceus.